The present invention relates to specific antibodies for use in preparing pharmaceutical compositions useful for the prevention or treatment of gastritis, gastric ulcers and duodenal ulcers caused by infection of Helicobacter pylori (hereinafter referred to as H. pylori or Hp), and for use as an additive to foods useful for the prevention of gastritis, gastric ulcers and duodenal ulcers.
At present it is believed that eradication of H. pylori in the stomach is essential for treating peptic ulcers fully. The combination of antibiotics and suppressors of gastric acid secretion has been generally proposed as a therapy for effective eradication of H. pylori as described below.
H. pylori is a gram-negative spiral rod-shaped bacterium having some flagella at one end and inhabiting the human gastric mucosa. Marshall, B. J. and Warren, J. R. in Australia reported in 1983 that this bacterium was frequently detected in stomach biopsy specimens from patients with gastric ulcers. At that time this bacterium was named Campylobacter pylori since it resembles Campylobacter in morphology and growth characteristics. Later, it was found that the bacterium is different from Campylobacter in the fatty acid composition of its outer membrane and sequence of ribosome 16S-RNA. Therefore, the bacterium is now referred to as Helicobacter pylori and belongs to a newly established genus of Helicobacter.
Since then, many reports have been published based on epidemiological studies, indicating that this bacterium causes gastritis, gastric ulcers and duodenal ulcers and is associated with diseases such as gastric cancer. Once H. pylori colonizes gastric mucosa, it cannot be eradicated in the stomach and continues to inhabit the stomach, although the immune response to infection thereof is strong, i.e., the antibody titer is high. Therefore, unless H. pylori is completely eliminated from the stomach by antibiotic therapy, the condition of infection will return to the same level as before treatment within about a month after the administration of antibiotics is stopped. Additionally, the pH of the stomach is maintained very low by HCl, which is a strong acid, and therefore most antibiotics are apt to be inactivated. For this reason, the combination of an antibiotic and a proton pump inhibitor which strongly suppresses the secretion of gastric acid is utilized often in a greater dose than usual for eradication of H. pylori. Recently, a new treatment employing a combination of bismuth subsalicylate, metronidazole, and tetracycline has proved to have the highest rate of elimination of H. pyroli, but metronidazole in this combination is known to cause the rapid emergence of an antibiotic-resistant strain when used alone. In developing countries this medicine has been used widely for treating diarrhea patients, and as a result there is a high rated infection with metronidazole-resistant H. pylori. Thus, the administration of antibiotics for a long time has the serious problems of increasing antibiotic-resistant strains as well as causing side effects.
At present, an immunological therapy approach using an oral vaccine has been proposed in order to solve problems such as side effects and increase of antibiotic-resistant strains by treatment with antibiotics for the eradication of the bacteria. However, for this purpose it is essential to develop model animals for Hp infection. It has been reported that Hp infection tests have been conducted using rats, mice, rabbits, dogs, pigs, and monkeys, and tests using dogs, pigs and monkeys have been successful. However, Hp cannot easily infect small animals such as mice and rats, and infection tests require complicated conditions. For example, germ-free animals are required for infection or fresh isolates are required for maintaining infection for a long time. These requirements have obstructed studies aimed at developing new methods for prevention and treatment.
For example, it was reported by Marchetti, M. et al. in Science, vol. 267, pp1655-1658 (1995) that 80% of Hp infection was inhibited by oral immunization using a mouse model for evaluating the efficacy of Hp oral vaccine. However, this oral vaccine preparation had heat-labile toxin (LT) derived from E. coli as an adjuvant. Generally. in such experiments using an oral vaccine, the vaccine preparation has cholera toxin in addition to LT derived from E. coli, and mucosal immunity cannot be attained without these adjuvants. LT from E. coli and cholera toxin have a high level of toxicity, and this vaccine method has many unsolved problems in respect to safety in its practical application to humans. Also, the vaccine is predominantly used for prevention, and therefore it has no effect on patients who have already been infected with Hp.
As a new attempt to inhibit Hp, Aiba et al. (The Meeting of the 30th Japan Germ-free Animal Gnotobiology Society, Program and Abstracts, pp22, Requested Title 18, New Attempt for Inhibiting Helicobacter pylori, January 1997) used germ-free mice as a model of Hp infection and studied (i) the effect of Lactobacillus salivarius as probiotics on inhibition of Hp and (ii) the effect of oral administration of anti-Hp antibodies obtained from the yolk of the eggs of hens immunized with formalin-killed whole cell of Hp on inhibition of Hp. In case (i), the number of Hp in the stomach of the Hp infected mice in an administered group became 10-1000 times less than that of a control group. In case (ii), the number of Hp in the stomach of an administered group became 10 times less than that of a control group.
However, these results were obtained using mice which do not have normal flora in the oral cavity, stomach and intestines, and it is not expected that such results can be obtained in conventional mice having normal flora. Generally, when mice having inherent normal flora are inoculated with human lactic acid bacteria, the bacteria are eliminated by the normal flora and do not colonize. Also, the antibodies used in the above experiments were those against whole cells of Hp, and the number of Hp in the stomach became only 10 times less than that of the control group, i.e., Hp was not eliminated completely. Furthermore, there is no reference to the relation between the decrease of Hp and the lessening of gastritis.
Japanese Patent Application Kokai No. 4-275232 also discloses the use of specific antibodies, but it merely discloses antibodies obtained from the eggs of hens immunized against Hp whole cells as an antigen. It describes a food for use in prevention of gastritis, gastric ulcer or duodenal ulcer, comprising as an active ingredient antibodies obtained from the eggs of hens immunized with Hp whole cell as an antigen, the antibodies being specific for the antigen, but the effect of the specific antibodies is not clear. The efficacy of the obtained antibodies against whole cells is evaluated by utilizing the fact that Hp adheres to mucin in pig gastric mucosa in vitro, and the results are that egg antibodies against Hp whole cells inhibit the adhesion of Hp to gastric mucosa. However, the in vitro tests were conducted in a mild environment of pH 7.4, and therefore it is questionable whether the obtained data accurately indicate the results to be obtained in a strongly acidic environment of pH 1-3 in the stomach. The confirmation of the effect of eliminating Hp in a strongly acidic stomach requires animals as an Hp infection model in experiments. The above-described patent application does not refer to such experiments, and therefore it is not clear whether the administration of egg antibodies against Hp whole cells can promote the elimination of Hp. Also, there is no reference as to whether the antibodies can suppress the occurrence of gastritis.
It is disclosed in transactions of Japan Agricultural Chemistry Society, 71, pp52, 20p22 (1997) that the egg antibodies against Hp whole cells have an inhibitory effect on Hp growth. However, the antibodies may have the same problems as explained above since the antigen used in immunizing hens is Hp whole cells.
It is known that antibodies against Hp whole cells can be obtained from milk or serum of mammalian animals such as cows. See, Japanese Patent Application Kokai No. 4-169539 and Japanese Patent Application Kokai No. 4-330099. These methods cannot produce antibodies in large amounts and inexpensively. In addition, the antigen used in immunization of mammals is whole cells of Hp, and therefore the complete elimination of Hp is not expected as mentioned in the above Aiba et al. prior art.
As explained above, the long-term use of antibiotics for elimination of Hp results in an increase in antibiotic-resistant bacteria as well as side effects, and a vaccine has not been developed for practical use. Also, attempts to administer lactic acid bacteria or to use egg antibodies against Hp whole cells cannot eradicate Hp, and therefore are not effective for prevention or treatment of gastritis, gastric ulcers and duodenal ulcers.
It is an object of the present invention to provide a pharmaceutical composition for use in preventing or treating gastritis, gastric ulcers and duodenal ulcers caused by H. pylori infection, the composition being effective and safe without the disadvantages of side effects and increase of drug-resistant strains, and to provide a food for preventing these diseases.
It is another object of the present invention to provide specific antibodies for use in preparation of the above pharmaceutical compositions.
Other objects and advantages as well as the nature of the present invention will be apparent from the following description.
The present inventors have found the mechanism of colonization of Hp to gastric mucosa, which mechanism has not been elucidated fully, and completed the present invention based on this discovery.
H. pylori cannot grow in an atmospheric condition, grows sub-optimally in an anaerobic condition, and grows optimally in a micro-aerobic condition. Due to this specificity, the ecology of the bacterium in the stomach has not been elucidated. Especially, the reason for strong growth ability in the stomach could not be explained, although various studies have been conducted on the colonization of Hp in gastric mucosa which is the key of growth in the stomach having strong acidity. The colonization of Hp in gastric mucosa plays an important role in the growth of Hp in the stomach, and therefore elucidation of colonization factors is of great significance in developing a method for preventing or treating gastritis, gastric ulcers and duodenal ulcers caused by Hp.
Pathogenic factors of Hp in the stomach have been considered to be urease produced by Hp, flagella for moving freely in the mucosal mucin layer, Cag A outer membrane protein involved in the production of interleukin 8 as an inflammatory cytokine, Vac A vacuolating cytotoxin concerned in vacuolation, erosion, necrosis and ulcer formation of gastric mucosal epithelial cells.
One of the pathogenic factors, urease produced by Hp, has been considered to be able to provide an environment useful for growth of Hp in the stomach by converting urea in the stomach to ammonia and neutralizing a strongly acidic environment around the bacteria cells. On the other hand, with respect to the colonization of Hp in the gastric mucosa, it has been shown that a urease-negative strain of Hp can colonize the stomach as well as a urease-positive strain in experiments using gnotobiotic pigs (Eaton, K. A. and Krakowka, S., 1995, Scand. J. Gastroenterol, 30:434-437). In a recent report, the binding of a urease-negative strain of Hp to human gastric mucosal epithelial cells and to cells from gastric adenocarcinoma cell line (Kato III cells) was compared with the binding of urease-positive strain, and it was concluded that there is no difference between adherence of the two kinds of strains to these cells and that the urease of Hp does not function as an adhesin. (Clyne, M. and Drumm, B. 1996, Infect. Immun. 64:2817-2820)
The present inventors have made the discovery, not expected from the results of prior art studies on the colonization of Hp, that urease itself participates in the colonization of Hp in the gastric mucosa. That is to say, the inventors have found that urease produced by Hp functions as a main adhesin in addition to functioning as an enzyme capable of converting urea to ammonia, and that the binding of urease itself to gastric mucosal mucin enables the growth of Hp. Also, the inventors obtained information suggesting that flagella of Hp also participate in the colonization of Hp in gastric mucosa.
The present invention was made based on the above discovery suggesting that antibodies against whole cells of Hp are not sufficient and antibodies against urease of Hp and/or flagella of Hp are effective for completely inhibiting the colonization of Hp in gastric mucosa to inhibit the growth of Hp in the stomach. It was further found that the combination of each or both of these antibodies and at least one organism selected from lactic acid bacteria, Enterococcuses, yeasts and Bacillus has a synergistic effect.
In one aspect, the present invention provides specific antibodies obtained from eggs laid by hens which have been immunized against urease of Helicobacter pylori as an antigen, the specific antibodies being active against the antigen. The present invention also provides specific antibodies obtained from eggs laid by hens which have been immunized against flagella of Helicobacter pylori as an antigen, the specific antibodies being active against the antigen.
The above anti-urease antibodies are active against urease, and the above anti-flagella antibodies are active against flagella. Therefore, anti-urease antibodies and/or anti-flagella antibodies are effective for inhibition of H. pylori growth in the stomach. The anti-urease antibodies can be used along with at least one organism selected from lactic acid bacteria, Enterococcuses, yeasts, and Bacillus in inhibiting H. pylori growth in the stomach. The anti-urease antibodies can be used along with the anti-flagella antibodies and at least one organism selected from lactic acid bacteria, Enterococcuses, yeasts, and Bacillus in inhibiting H. pylori growth in the stomach.
In another aspect, the present invention provides a pharmaceutical composition containing as an active ingredient the above-described anti-urease antibodies and/or the above-described anti-flagella antibodies for use in preventing or treating gastritis, gastric ulcer and duodenal ulcer. The anti-urease antibodies can be used along with at least one organism selected from lactic acid bacteria, Enterococcuses, yeasts, and Bacillus in prevention or treatment of gastritis, gastric ulcers and duodenal ulcers. The anti-urease antibodies can be used along with the anti-flagella antibodies and at least one organism selected from lactic acid bacteria, Enterococcuses, yeasts, and Bacillus in prevention or treatment of gastritis, gastric ulcers, and duodenal ulcers.
The present invention also provides a food containing as an additive the above-described anti-urease antibodies and/or the above-described anti-flagella antibodies for use in preventing gastritis, gastric ulcers and duodenal ulcers. To a food containing the anti-urease antibodies, or to a food containing the anti-urease antibodies and the anti-flagella antibodies, at least one organism selected from lactic acid bacteria, Enterococcuses, yeasts, and Bacillus can be added.